Hydrogen sulfide (H 2 S) is emerging as an important gasotransmitter in both physiological and pathological states. Rapid measurement of H 2 S remains a challenge. We report a microfluidic method for rapid measurement of sulphide in blood plasma using Dansyl-Azide, a fluorescence (FL) based probe. We have measured known quantities of externally added (exogenous) H 2 S to both buffer and human blood plasma. Surprisingly, a decrease in FL intensity with increase in exogenous sulphide concentration in plasma was observed which is attributed to the interaction between the proteins and sulphide present in plasma underpinning our observation. The effects of mixing and incubation time, pH, and dilution of plasma on the FL intensity is studied which revealed that the FL assay required a mixing time of 2 min, incubation time of 5 min, a pH of 7.1 and performing the test within 10 min of sampling; these together constitute the optimal parameters at room temperature. A linear correlation (with R 2 ≥ 0.95) and an excellent match was obtained when a comparison was done between the proposed microfluidic and conventional spectrofluorometric methods for known concentrations of H 2 S (range 0–100 µM). We have measured the baseline level of endogenous H 2 S in healthy volunteers which was found to lie in the range of 70 μM – 125 μM. The proposed microfluidic device with DNS-Az probe enables rapid and accurate estimation of a key gasotransmitter H 2 S in plasma in conditions closely mimicking real time clinical setting. The availability of this device as at the point of care, will help in understanding the role of H 2 S in health and disease. © 2019, The Author(s).